A pre-requisite for applying the helper strain concept for fungal genome modification is the ability to form a heterokaryon by fusing two different cell types: the helper strain (HS) and the target strain (TS). In some fungal cells (e.g., N. crassa), this fusion can be accomplished by simply mixing the spores of the HS and TS and allowing them to germinate together on selective medium. In other fungal cells (e.g., T. reesei), cell fusion does not occur so readily, and it is necessary to make protoplasts to facilitate fusion. Once the cells from the HS and TS are fused, the shared cytoplasm allows the benefit that the HS provides to be available to the TS. In N. crassa, the cells are connected into a syncytium which allows the cytoplasm as well as organelles to migrate between cell compartments (Roper, M. Simonin, A., Hickey, P. C., Leeder, A., and Glass, N. L. 2013. Nuclear dynamics in fungal chimera. Proc. Nat. Acad. Sci. 110 (32), 12875-12880). The beneficial components produced by the HS nuclei are, therefore, freely distributed throughout the mycelium. The compartments of T. reesei, on the other hand, are separated by septae that limit the migration of cell components.
Once the benefit from the heterokaryon is utilized, the component strains need to be separated. This is achieved by sporulating the heterokaryon, plating the individual conidiospores, and verifying strains that derived from single uninucleate spores. In N. crassa microconidia, containing a single nucleus, can be produced on media containing iodoacetate (Ebbole, D. and Sachs, M. S. 1990. A rapid and simple method for isolation of Neurospora crassa homokaryons using microconidia. Fungal Genet. Newslett. 37), while some T. reesei strains have predominantly multinucleate conidiospores. In T. reesei, if a strain that produces multinucleate conidiospores cannot be substituted with one that produces uninucleate conidiospores, additional rounds of spore-purification need to be performed, lengthening the procedure.
As a result of these and other differences, the helper strain concept has not been incorporated into methods for routine manipulation and strain improvement in T. reesei. 
Thus, there still remains a need for developing efficient and effective helper strain-mediated genome engineering methods and compositions for many fungal host cells, including T. reesei. 